DNA topoisomerases are essential enzymes which have critical roles in all aspects of chromosome functions including genome stability, transcription, chromosome replication and segregation. They are unique biochemical machines which can pass one DNA segment through another via a proteinmediated DNA gate. DNA topoisomerases are molecular targets of a number of clinically important antibiotics and anti-neoplastic agents. The mechanistic studies of topoisomerases thus provide useful information on the biochemical basis of cancer chemotherapy. Both these drugs and the drug-resistant topoisomerase mutants are important tools for analyzing the functions of these enzymes. The long-term goal of our research is on the structure, function, and mechanism of eucaryotic DNA topoisomerases. Our current work will focus on four specific aims. 1. We will develop protein foot-printing and single molecule fluorescence microscopy to study the mechanism of topoisomerase II. The biochemical mechanism of drug resistance will be addressed with these approaches. 2. While topo Ill isozymes have important biological functions in recombination/repair, the biochemical basis of their in vivo roles remains to be elucidated. We will use a plasmid DNA with a single stranded loop as a substrate to investigate the biochemical reactions of topoisomerase III, including cleavage and strand annealing/swiveling reactions. These biochemical properties may have important relevance to their in vivo functions. 3. The biological functions of topoisomerase III will be approached by using the genetic mutants isolated and characterized in our lab. The intracellular localization of these enzymes during Drosophila development will be examined. 4. The interactions between the N-terminal domain of topoisomerase I and the C-terminal repeats in the largest subunit of RNA polymerase II will be studied. This binding affinity of topoisomerase I may be important for its targeting to the transcriptionally active loci in chromatin.